Ether phospholipids are analogues of the naturally occurring 2-lyso-phosphatidylcholine that have been reported to have in vitro/in vivo antitumor activity. Their antiproliferative effect has been found against a variety of animal and human tumor cell lines. We have characterized the cytostatic activity of two ether phospholipids, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine and 1-hexadecylmercapto-2-methoxymethyl-rac-glycero-3-phosphocholine, on a human tumor cell line derived from a colon adenocarcinoma, HT29. We have used three different flow cytometric approaches to study which phase of the cell cycle was the most sensitive to the antiproliferative activity of the two ether phospholipids. By applying the biparametric analysis, 5'-bromo-2-deoxyuridine incorporation versus DNA content, we have been able to demonstrate that ether phospholipids do not interfere with the S phase of the cell cycle. The simultaneous measurement of total nuclear protein versus DNA content in isolated HT29 nuclei has enabled us to exclude a block in the M phase of the cell cycle. Finally, the stathmokinetic analysis has allowed us to show that the cytostatic activity of the two ether phospholipids 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine and 1-hexadecylmercapto-2-methoxymethyl-rac-glycero-3-phosphocholine is characterized by multiple "terminal points" as the drugs action results in a G1 block and in a slow-down of the transition from late S to G2, followed by an accumulation of HT29 cells in the G2 phase of the cell cycle.